Ethanol versus methanol for CD marker fixation RE: [Histonet] immunostaining post-FACS

From:Gayle Callis

The reason I answered Mark Tarango's email in the first place was to 
correct which alcohol I do use with acetone (100% ethanol).

It is known that methanol is not optimal for all antigens, and we avoid it 
with our murine CD markers.  It has been noted in the literature that CD 
marker staining may be compromised by fixation with methanol and certainly 
true of our murine CD markers (Elias book, the book is at home).  This is 
probably due to the hydrolysis of the protein antigen by methanol, and 
probably happens with ethanol too.

  There was a  Histonet discussion some years back (in Histonet archives) 
on the use of methanol for CD marker fixation, and it was noted by some 
that loss of staining after methanol did occur for some CD markers they 
were working with.   Human CD4 and CD8 do not work after ethanol fixation, 
and probably will be compromised badly by the acetone/ethanol mixture.  I 
believe Dr. Chris van der Loos in The Netherlands tried this.  He uses 
acetone for those markers

  We recently did a little inhouse study to compare the effects of 
methanol, acetone, acetone/absolute ethanol mixture for immunofluorescent 
staining of  cell cultures infected with a bacteria.  In this case, we had 
less fluorescence with methanol than with acetone, and had even better 
staining acetone/absolute ethanol  compared to acetone. The point is that 
doing a fixation panel is always wise, particularly when a new protocol is 
being set up - different fixatives and times with those 
fixatives).    Human CD4 and CD8 do not stain after ethanol fixation ( a 
fact Dr. Chris van der Loos brings up frequently, and consequently these 
human markers will be compromised by the acetone/ethanol mixture.  I 
believe Dr. Chris van der Loos in The Netherlands tried this and continues 
to use acetone for those markers.

As long as the fixation method works for you, then you may not want to 
change or try anything different.  Question:  have you ever tried another 
fixative as a comparison?  If you do, let us know the results.

At 10:40 PM 8/9/2007, you wrote:
>Hi Gayle,
>It's quite interesting that on the contrary we have a very good experience
>with methanol as a fixative for variety of antigens (immuno on
>cytospins)including CD's and nuclear antigens. Fixation in methanol
>preserves good cell morphology and yield a reliable (repeatable)
>immunoreactions confirmed by flow cytometry.
>I'm just trying to figure out where is the reason for such a difference?

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610

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